Synthetic methylmalonyl-CoA mutase transgene for the treatment of MUT class methylmalonic acidemia (MMA)

Abstract

Synthetic polynucleotides encoding human methylmalonyl-CoA mutase (synMUT) and exhibiting augmented expression in cell culture and/or in a subject are described herein. An adeno-associated viral (AAV) gene therapy vector encoding synMUT under the control of a liver-specific promoter (AAV2/8-HCR-hAAT-synMUT-RBG) successfully rescued the neonatal lethal phenotype displayed by methylmalonyl-CoA mutase-deficient mice, lowered circulating methylmalonic acid levels in the treated animals, and resulted in prolonged hepatic expression of the product of synMUT transgene in vivo, human methylmalonyl-CoA mutase (MUT).

The invention claimed is: 
     
       1. A method of treating a disease or condition mediated by methylmalonyl-CoA mutase, comprising administering to a subject in need thereof, in vivo or by genetic alteration of cells prior to administration, a therapeutic amount of a synthetic methylmalonyl-CoA mutase (MUT) polynucleotide (synMUT) selected from the group consisting of:
 a) a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:1; 
 b) a codon optimized polynucleotide comprising a polynucleotide having a nucleic acid sequence with at least about 70% identity to the nucleic acid sequence of SEQ ID NO:1 and encoding a polypeptide according to SEQ ID NO:2, and having equivalent expression in a host to either SEQ ID NO:1 expression or SEQ ID NO:3 expression, wherein the codon optimized polynucleotide having at least about 70% identity to SEQ ID NO:1 does not have the nucleic acid sequence of SEQ ID NO:3. 
 
     
     
       2. The method of  claim 1 , wherein the disease or condition is methylmalonic acidemia (MMA). 
     
     
       3. The method of  claim 1 , wherein the synthetic polynucleotide is formulated with a pharmaceutically acceptable carrier. 
     
     
       4. The method of  claim 1 , wherein the synthetic polynucleotide of  claim 1  is comprised within a viral vector selected from the group consisting of an adenoviral vector, a retroviral vector, a lentiviral vector, and an adeno-associated viral vector. 
     
     
       5. The method of  claim 1 , wherein the synthetic polynucleotide of  claim 1  is placed under the transcriptional control of a ubiquitous promoter, a tissue-specific promoter or a regulable promotor. 
     
     
       6. The method of  claim 1 , wherein the synthetic polynucleotide of  claim 1  is comprised within an expression cassette wherein the expression cassette comprises a promotor, the synthetic polynucleotide, and a polyadenylation signal. 
     
     
       7. The method of  claim 1 , wherein the synthetic polynucleotide of  claim 1  is comprised within a non-viral vector selected from the group consisting of naked DNA, mini-circules, liposomes, ligand-polylysine-DNA complexes, nanoparticles, cationic polymers, synthetic peptide complexes, artificial chromosomes and polydispersed polymers. 
     
     
       8. The method of  claim 1 , wherein the synthetic polynucleotide of  claim 1  is placed under the transcriptional control of a tissue-specific promoter selected from the group consisting of Apo A-I promoter, ApoE promoter, hAAT promoter, transthyretin promoter, liver-enriched activator, albumin promoter, PEPCK promoter, RNAP II  promoter, PAI-1 promoter, ICAM-2 promoter, MCK promoter, SMC α-actin promoter, myosin heavy-chain promotor, myosin light-chain promotor, cytokeratin 18 promoter, CFTR promoter, GFAP promoter, NSE promoter, Synapsin I promoter, Preproenkephalin promoter, dβH promoter, prolactin promoter, myelin basic protein promoter, ankyrin promoter, α-spectrin promoter, globin promoter, HLA-DRα promoter, CD4 promoter, glucose 6-phosphatase promoter, and dectin-2 promoter. 
     
     
       9. The method of  claim 1 , wherein the therapeutic amount of a synthetic methylmalonyl-CoA mutase (MUT) polynucleotide (synMUT) is delivered by a route of delivery selected from the group consisting of intradermal injection, subcutaneous injection, intravenous injection, intraperitoneal injection, intraocular injection, subretinal injection, renal artery injection, hepatic vein injection, intramuscular injection, ultrasound(-mediated transfection), electric field-induced molecular vibration, electroporation, transfection using laser irradiation, photochemical transfection, gene gun particle bombardment, parenteral administration and oral administration. 
     
     
       10. The method of  claim 4 , wherein the viral vector is AAV2/8-HCR-hAAT-RBG. 
     
     
       11. The method of  claim 1 , wherein the synthetic polynucleotide of  claim 1  is inserted into a cell of the subject via genome editing on the cell of the subject using a nuclease selected from the group of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPER/cas system) and meganuclease re-engineered homing endonuclease. 
     
     
       12. The method of  claim 1 , wherein the synthetic polynucleotide of  claim 1  has at least about 80% identity to the nucleic acid sequence of SEQ ID NO:1. 
     
     
       13. The method of  claim 1 , wherein the synthetic polynucleotide of  claim 1  has at least about 90% identity to the nucleic acid sequence of SEQ ID NO:1. 
     
     
       14. The method of  claim 1 , wherein the synthetic polynucleotide of  claim 1  has at least about 95% identity to the nucleic acid sequence of SEQ ID NO:1. 
     
     
       15. A method of treating a disease or condition mediated by methylmalonyl-CoA mutase, comprising administering to a subject in need thereof a therapeutic amount of a mature methylmalonyl-CoA mutase produced using a synthetic methylmalonyl-CoA mutase (MUT) polynucleotide (synMUT) selected from the group consisting of: (a) a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:1; and (b) a codon-optimized polynucleotide comprising a polynucleotide having a nucleic acid sequence with at least about 70% identity to the nucleic acid sequence of SEQ ID NO: 1 and encoding a polypeptide comprising residues 33-750 of SEQ ID NO:2, and having equivalent expression in a host to either SEQ ID NO: 1 expression or SEQ ID NO:3 expression, wherein the codon-optimized polynucleotide having at least about 70% identity to SEQ ID NO:1 does not have the nucleic acid sequence of SEQ ID NO:3. 
     
     
       16. The method of  claim 15 , wherein the disease or condition is methylmalonic academia (MMA). 
     
     
       17. The method of  claim 15 , wherein the mature methylmalonyl-CoA mutase polypeptide is formulated with a pharmaceutically acceptable carrier. 
     
     
       18. A synthetic methylmalonyl-CoA mutase (MUT) polynucleotide (synMUT) comprising a nucleic acid sequence with at least 70% identity to the nucleic acid sequence of SEQ ID NO:1, encoding a polypeptide according to SEQ ID NO:2, and having equivalent expression in a host to either SEQ ID NO:1 expression or SEQ ID NO:3 expression, wherein the synthetic polynucleotide does not have the nucleic acid sequence of SEQ ID NO:3. 
     
     
       19. The synthetic polynucleotide of  claim 18 , wherein the synthetic polynucleotide exhibits increased expression in an appropriate host relative to the expression SEQ ID NO:3 in an appropriate host. 
     
     
       20. The synthetic polynucleotide of  claim 18 , wherein the synthetic polynucleotide comprises a nucleic acid sequence comprising codons that have been optimized relative to the naturally occurring human methylmalonyl-CoA mutase polynucleotide sequence (SEQ ID NO:3). 
     
     
       21. The synthetic polynucleotide of  claim 18 , wherein the synthetic polynucleotide is comprised within a viral vector selected from the group consisting of an adenoviral vector, a baculoviral vector, a retroviral vector, a lentiviral vector, a foamy viral vector, a herpes viral vector, a Moloney murine leukemia viral vector, a Vaccinia viral vector, a hepatitis viral vector, and an adeno-associated viral vector. 
     
     
       22. The synthetic polynucleotide of  claim 18 , wherein the synthetic polynucleotide of  claim 1  is comprised within a non-viral vector selected from the group consisting of naked DNA, mini-circules, liposomes, ligand-polylysine-DNA complexes, nanoparticles, cationic polymers, synthetic peptide complexes, artificial chromosomes and polydispersed polymers. 
     
     
       23. The synthetic polynucleotide of  claim 18 , wherein the synthetic polynucleotide is comprised within an expression cassette wherein the expression cassette comprises a promotor, the synthetic polynucleotide, and a polyadenylation signal. 
     
     
       24. The synthetic polynucleotide of  claim 23 , wherein the expression cassette further comprises a 5′ intron. 
     
     
       25. The synthetic polynucleotide of  claim 23 , wherein the expression cassette further comprises an mRNA stability element. 
     
     
       26. The synthetic polynucleotide of  claim 25 , wherein the mRNA stability element is the woodchuck post-transcriptional regulatory element. 
     
     
       27. The synthetic polynucleotide of  claim 23 , wherein the expression cassette further comprises a hepatic control region. 
     
     
       28. The synthetic polynucleotide of  claim 23 , wherein the expression cassette further comprises an inverted terminal repeat. 
     
     
       29. The synthetic polynucleotide of  claim 18 , wherein the synthetic polynucleotide is under the transcriptional control of a ubiquitous promoter, a tissue-specific promoter or a regulable promotor. 
     
     
       30. The synthetic polynucleotide of  claim 29 , wherein the synthetic polynucleotide is under transcriptional control of a tissue-specific promoter selected from the group consisting of Apo A-I promoter, ApoE promoter, hAAT promoter, transthyretin promoter, liver-enriched activator, albumin promoter, PEPCK promoter, RNAP II  promoter, PAI-1 promoter, ICAM-2 promoter, MCK promoter, SMC α-actin promoter, myosin heavy-chain promotor, myosin light-chain promotor, cytokeratin 18 promoter, CFTR promoter, GFAP promoter, NSE promoter, Synapsin I promoter, Preproenkephalin promoter, dβH promoter, prolactin promoter, myelin basic protein promoter, ankyrin promoter, α-spectrin promoter, globin promoter, HLA-DRα promoter, CD4 promoter, glucose 6-phosphatase promoter, and dectin-2 promoter. 
     
     
       31. The synthetic polynucleotide of  claim 29 , wherein the synthetic polynucleotide is under transcriptional control of a viral promoter selected from the group consisting of the ubiquitous cytomegalovirus immediate early (CMV-IE) promoter, the chicken beta-actin (CBA) promoter, the simian virus 40 (SV40) promoter, the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter, and the Moloney murine leukemia virus (MoMLV) LTR promoter. 
     
     
       32. An expression vector comprising the synthetic polynucleotide of  claim 18 . 
     
     
       33. The expression vector of  claim 32 , wherein the expression vector is AAV2/8-HCR-hAAT-RBG.